Qiong Wu & Mengji Cao & Huanan Su & Sagheer Atta & Fangyun Yang & Xuefeng Wang & Changyong Zhou

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1 DOI /s Molecular characterization and phylogenetic analysis of itrus viroid I-LSS variants from citrus in Pakistan and hina reveals their possible geographic origin Qiong Wu & Mengji ao & Huanan Su & Sagheer tta & Fangyun Yang & Xuefeng Wang & hangyong Zhou ccepted: 2 January 2014 # KNPV 2014 bstract Vd-I-LSS (low sequence similarity), a variant of itrus bent leaf viroid (BLVd), was first discovered in Japan, and its distribution is currently limited to Japan and Iran. In the present study, seven Vd-I-LSS isolates were detected from different citrus hosts (itrus sinensis,. reticulata and. limettioides) in Pakistan and hina. enetic diversity analysis of 49 cdns of Vd-I-LSS isolates showed that the Pakistan population was more diverse than that tested from Japan or hina. Phylogenetic analysis clustered the predominant sequences examined into three main clades. Only sequences from the Pakistan isolates were found in all three clades, suggesting Pakistan may be the original source of Vd-I-LSS. ultivar import records and the close phylogenetic relationship Qiong Wu and Mengji ao are equal contributors. Q. Wu: M. ao : H. Su : F. Yang : X. Wang (*) :. Zhou (*) National itrus Engineering Research enter, itrus Research Institute, Southwest niversity, hongqing , People s Republicofhina wangxuefeng@cric.cn zhoucy@cric.cn Q. Wu: H. Su ollege of Plant Protection, Southwest niversity, hongqing , People s Republicofhina S. tta ollege of griculture Dera hazi Khan, niversity of griculture, Faisalabad, Pakistan found between Vd-I-LSS from hina and Japan suggested that the viroid isolated from hina might have originated from Japan. Keywords Vd-I-LSS. itrus bent leaf viroid. enetic diversity. Phylogenetic analysis Viroids are covalently closed, single-stranded, circular RNs of nucleotides (nt) that are the smallest pathogens known. itrus trees are natural hosts of at least seven viroid species of the family Pospiviroidae: itrus exocortis viroid (EVd), itrus bent leaf viroid (BLVd), Hop stunt viroid (HSVd), itrus bark cracking viroid (BVd), itrus dwarfing viroid (DVd), itrus viroid V (Vd-V) and itrus viroid VI (Vd-VI) (Duran-Vila et al. 1988b; Ito et al. 2001; Serra et al. 2008). Two variants of BLVd, in the genus pscaviroid, were previously recognized. These were designated as itrus viroid Ia (Vd-Ia) (327~329 nt) and Vd-Ib (315~319 nt) and distinguished by their mobility in sequential polyacrylamide gel electrophoresis (spe) (Duran-Vila et al. 1988a; shulin et al. 1991). new variant of BLVd was discovered in 2000 (Ito et al. 2000) and named Vd-I-LSS (low sequence similarity). Vd-I-LSS induces similar symptoms on Etrog citron (itrus medica L.) as those reported for BLVd variants, with the exception of a slightly more severe epinasty (Ito et al. 2000). Sequence analyses of Vd-I-LSS variants consisting of 325~330 nt showed only 82 %~85 %

2 sequence similarity to BLVd, which was significantly lower than the 93 %~100 % sequence similarities among BLVd variants (Vd-Ia and Vd-Ib). Most of the nucleotide changes between the Vd-I-LSS and BLVd variants were located at complementary positions of the upper and lower strands of the predicted rod-like secondary structure, more specifically within the pathogenic (P), variable (V), and right terminal (T R ) domains, as well as around the central conserved region (R). Since Vd-I-LSS was first discovered in Japan, this variant has been reported additionally only in Iran (Ito et al. 2000; lavi et al. 2005). Though citrus samples imported from Pakistan to Japan were confirmed to be infected with Vd-I-LSS at post-entry quarantine (Inoue et al. 2003), the occurrence of the Vd-I-LSS has never been reported in Pakistan in which other viroid species, including EVd, BLVd, HSVd, DVd and Vd-V, have been identified (rif et al. 2005; Mughal 2004; ao et al. 2009, 2013). In hina, many mandarins or mandarin hybrids have been imported from Japan. However, information on the existence of Vd-I-LSS in these introductions is still pending. In this study, Vd-I-LSS was identified and characterized from citrus samples collected from hina and Pakistan. total of 50 viroid-infected trees were sampled from six hinese Provinces including Zhejiang, Sichuan, Jiangxi, Hunan, Yunnan and hongqing, and from the Punjab Province in Pakistan (ao et al. 2010, 2013). Total nucleic acids were extracted as previously described (Zhou et al. 2001). One-step RT-PR assay (Wang et al. 2008) using a proof-reading thermostable DN polymerase was conducted using reverse primer Vd-I-LSS-R (5 -TTTTT-3 complementary to position 75 94) and forward primer Vd-I-LSS-F (5 -TTT T-3 homologous to position ) to obtain full length Vd-I-LSS (Ito et al. 2000). Due to the relatively high similarity and risk of false positives, the Vd-Ia and Vd-Ib variants were also detected with two primer sets B2-TM (5 -TTT-3 complementary to position 315 7) and B2-TP (5 -TTTT TTTTTT-3 homologous to position 8 27), and B2-M (5 -TTTTT-3 complementary to position 76 95) and B2-P (5 -TTTT-3 homologous to position ) (Ito et al. 2000). RT-PR products were electrophoresed in 2 % agarose gels and visualized by ethidium bromide staining under V light (VI Innotech). The RT-PR products were purified with the PR purification kit (Promega), and the resulting fragments were cloned into pmd19-t vectors (TaKaRa) and transformed into Escherichia coli DH5α cells (TaKaRa). Six to eight clones were randomly selected and sent to the Beijing enomics Institute (BI) for sequencing. Multiple sequences were aligned using the lustal W Program (Thompson et al. 1994). The phylogenetic tree was constructed following the neighbourjoining method tested with 1,000 bootstrap replications (Tamura et al. 2011). Pairwise-deletion was selected to minimize the effect caused by insertion/ deletion through alignment. enetic distance analyses were conducted using the Jukes-antor Model (Jukes and antor 1969) with the ME 5.0 software (Tamura et al. 2011). HSVd sequence (N001351) was used as an outgroup for phylogenetic analysis. The secondary structures were predicted at a folding temperature of 25 using the online mfold web server ( Form2.3) and subsequently modified with the RNviz program (De Rijk et al. 2003). Seven samples of unknown sweet orange (. sinensis), Succri sweet orange, Sweet lime (. limettioides), Red blood sweet orange, Kinnow mandarin (. reticulata)in Pakistan, and two hinese mandarin hybrids, kemi and Nishirkaori, were found to be infected with Vd-I-LSS (Fig. 1, Table 1). The analysis also showed the presence of two other BLVd variants (Vd-Ia and Vd-Ib) (Table 1). Forty-nine independent cdn clones of the seven Vd-I-LSS isolates were sequenced. The genome Fig. 1 One step RT-PR detection of Vd-I-LSS. Lane M: DN marker with 100 bp ladder; lane P: positive control; lane N: negative control; lane W: water control; lanes 1~7: Vd-I-LSS isolates from different samples: unknown sweet orange (SO), Succri sweet orange (S), Sweet lime (SL), Red blood sweet orange (RB), Kinnow mandarin (KM) in Pakistan and kemi (K), and Nishirkaori (NI) in hina respectively

3 Table 1 RT-PR analysis of three BLVd variants in samples from different origin and host Isolate Host Origin Vd-I-LSS Vd-Ia Vd-Ib SO Sweet orange Punjab, Pakistan + S Succri Punjab, Pakistan + SL Sweet lime Punjab, Pakistan + RB Red blood sweet orange Punjab, Pakistan + + KM Kinnow mandarin Punjab, Pakistan + + K kemi Zhejiang, hina + NI Nishirkaori Zhejiang, hina + + sizes of the different isolates varied from 323 to 329 nt with 86 % falling within the range of 325~330 nt as reported previously, while six clones from isolate SO were smaller than expected (323 nt). Vd-I-LSS sequences shared % identity with Vd-I-LSS sequences deposited in NBI and only % with Vd-Ia and Vd-Ib. The sequence variant that occurred twice or more times in the same isolate was considered the predominant sequence. The genetic diversity of the five Vd-I-LSS isolates from Pakistan and two isolates from hina was assessed along with eight reported Vd-I-LSS sequences from Japan, giving a total of 57 sequences (Table 2). ll these sequences were aligned and resulted in a total of 335 positions. mong the 43 variable sites found, 34 were parsimoniously informative (two or more clones had the same mutation) and the remaining nine sites were singletons. The nucleotide diversity estimated from the nucleotide distances of all Vd-I-LSS populations was 0.036±0.006, which was close to that determined for the population from Pakistan (0.038±0.007). The nucleotide diversity of the populations from hina and Japan were lower, 0.030±0.006 and 0.026±0.005, respectively. Table 2 Nucleotide diversity of Vd-I-LSS isolates No. of isolates No. of clones sequenced V Pi S N Pakistan ±0.007 hina ±0.006 Japan a ±0.005 ll ±0.006 conserved sites; V variable sites; Pi parsimonious informative nucleotide sites; S singleton sites; N nucleotide diversity a unknown Phylogenetic analysis of nine predominant Vd-I- LSS sequences from the seven isolates sequenced in this study and eight nucleotide sequences from Japan deposited in enbank showed that all sequences were clustered into three main clades (Fig. 2). It is worth noting that Pakistan sequences were distributed in all three clades, and the SO-1 clone from Pakistan with the smallest size (323 nt) was the only component of clade II. lade III contained KM-1, WS1 and WS2 isolates from Japan. These isolates all had 15 nucleotide substitutions and one insertion at site 259 (Δ ) compared to the reference sequence. The remaining 13 sequences, including five from Pakistan, two from hina and six from Japan, were clustered in clade I. Two isolates (K, NI) from Zhejiang, hina clustered into sub-clade. onsidering the cultivar import records from Japan to hina and data from this study, the Vd-I-LSS isolated from Zhejiang Province (kemi and Nishirkaori) is thought to have originated from Japan. Based on genetic and phylogenetic analyses and export of infected materials (Inoue et al. 2003), Pakistan might be an important source of Vd-I-LSS although the original source of Vd-I-LSS remains uncertain. Sequence alignments showed that the numbers of sequence variations were four in sub-cluster, two in sub-cluster B, four in sub-cluster, seven in sub-cluster D, eight in sub-cluster E, five in sub-cluster F, 17 in subcluster and 22 in sub-cluster H, compared with the Vd-I-LSS reference sequence (N ). The predicted secondary structure showed that sequence variations occurred mainly in the P domain. In addition, the T R and T L regions were also at the high probability of variation (Fig. 3). Notably, a transition (234 ) of SO-5 sequence was found in the lower strand of the R, which consists of 16 and 17 core nucleotides in the upper and lower strands, respectively, and is considered to be the most conserved region within the genus

4 VdI-LSS-ref (N004358) 83 VdI-LSS (B019509) JVd2 (E36218) JVd2 (E36217) VdI-LSS-SL-1(KF726091) 84 VdI-LSS-SL-4(KF726092) VdI-LSS-K-4(KF726093) B VdI-LSS-NI-1(KF726099) VdI-LSS-TS (B054638) D 60 VdI-LSS-9K-S-(B054641) VdI-LSS-SO-5(KF726094) VdI-LSS-RB-1(KF726095) VdI-LSS-S-4(KF726096) E F VdI-LSS-SO-1(KF726098) VdI-LSS-WS1 (B054639) VdI-LSS-KM-1(KF726097) VdI-LSS-WS2 (B054640) HSVd-ref (N001351) H lade I lade II lade III 0.1 Fig. 2 Neighbour-joining tree of Vd-I-LSS isolates. ll Vd-I- LSS sequences were divided into three main clades (I, II and III). Numbers on the nodes represent bootstrap values (%) based on 1,000 replicates. Branches with bootstrap support less than 60 % pscaviroid (Fig. 3 E). Transition of 274 ( ) and transversion of 276 ( ) in variants of SL-1 and SL-4 from Pakistan in sub-clade B extended the base pairing were collapsed. Samples with solid and empty triangles indicate those from Pakistan and hina, respectively, and samples unlabeled came from Japan. HSVd sequence was used as an outgroup. enbank accession numbers were listed in the parentheses and disabled the secondary structure to form a loop (Fig. 3). Substitutions of 275 ( ) and 276 ( ) resulted in a smaller loop of the rod-like secondary TL P V TR B D E F H Fig. 3 Proposed secondary structure of Vd-I-LSS of minimum free energy. Rectangular boxes show the nucleotide mutations in the five putative domains. Letters refer to those in Fig. 2, indicating different clades or subclades

5 structure among all the variants in the sub-clusters, D, and E (Fig. 3). nother substitution in the left terminal region (318 ) was found in sub-cluster D, E and (Fig. 3). In addition to the transitions at all three sites (274, 275, 276 ), four more substitutions, one in the upper strand (65 ) and three in the lower strand (246, 279, 84 ), occurred in the P domain of sub-clades and H (Fig. 3), which contained WS1 from Japan and KM, SO from Pakistan. In conclusion, Vd-I-LSS isolates were detected and identified from different citrus cultivars in Pakistan and hina. This finding significantly expands the known geographic distribution of Vd-I-LSS and provides new genomic information relative to putative routes of pathogen entry. Further biological characterization with infectious clones of Vd-I-LSS, as well as additional surveys in more regions would be helpful for understanding the classification and origin of Vd-I-LSS. cknowledgments This work was supported in part by Special Fund for gro-scientific Research in the Public Interest ( ), National Natural Science Foundation of hina ( ) and MO s Program for an Outstanding Scientist and Innovative Research Team. We wish to acknowledge Jin Zhang for her help with plant materials. We thank Drs E. iverolo and R. Yokomi for critical reading and editing the manuscript. References lavi, S. M., Sohi, H. H., hoonmanesh,., Rahimian, H., & Barzegar,. (2005). Molecular characterization and identification of multiple viroids in mazandaran province. Iranian Journal of Plant Pathology, 41(2), rif, M., hmad,., Ibrahim, M., & Hassan, S. (2005). Occurrence and distribution of virus and virus-like diseases of citrus in north-west frontier province of Pakistan. Pakistan Journal of Botany, 37(2), 407. shulin, L., Lachman, O., Hadas, R., & Barjoseph, M. (1991). Nucleotide sequence of a new viroid species, itrus bent leaf viroid (BLVd) isolated from grapefruit in Israel. Nucleic cids Research, 19(17), ao, M. J., tta, S., Liu, Y. Q., Wang, X. F., Zhou,. Y., Mustafa,., et al. (2009). First report of itrus bent leaf viroid and itrus dwarfing viroid from itrus in Punjab, Pakistan. Plant Disease, 93(8), 840. ao, M. J., Liu, Y. Q., Wang, X. F., Yang, F. Y., & Zhou,. Y. (2010). First report of itrus bark cracking viroid and itrus viroid V infecting citrus in hina. Plant Disease, 94(7), 922. ao, M. J., Su, H. N., tta, S., Wang, X. F., Wu, Q., Li, Z.., et al. (2013). Molecular characterization and phylogenetic analysis of itrus viroid V isolates from Pakistan. European Journal of Plant Pathology, 135(1), De Rijk, P., Wuyts, J., & De Wachter, R. (2003). RnaViz 2: an improved representation of RN secondary structure. 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Viroids are unique plant pathogens that are smaller than viruses and consist of a short single-stranded circular RNA without a protein coat (Diener,

Citrus Viroids Viroids are unique plant pathogens that are smaller than viruses and consist of a short single-stranded circular RNA without a protein coat (Diener, 1971). These pathogens cause damage to